Posttranslational modifications of proteins are required for many cellular functions including the mediation of protein-protein interactions, enzymatic activity, protein degradation, localization of proteins to cellular compartments and maintenance of protein stability. Ubiquitination, one type of protein modification, occurs when the protein ubiquitin becomes ligated to a target protein. The attachment of one or more ubiquitin molecules most often triggers the destruction of the protein substrate but has recently been shown to direct further trafficking of the protein. The process of ubiquitination involves the concerted activity of several enzymes that comprise the ubiquitin complex (UbC). The three major enzymes of the complex include a ubiquitin-activating enzyme (E1) which activates ubiquitin using ATP, a ubiquitin-conjugating enzyme (E2) which receives the activated ubiquitin and can transfer the molecule to the protein recipient, and a ubiquitin-ligase enzyme (E3) which plays the crucial role of determining the specificity of ubiquitination (Yamao, J. Biochem. (Tokyo), 1999, 125, 223-229).
Recently, Pirozzi et al. identified two novel E3 ubiquitin ligase enzymes, WWP1 and WWP2, each of which contain WW domains comprising a region of 38-40 amino acids which includes two highly conserved tryptophans and an invariant proline (Pirozzi et al., J. Biol. Chem., 1997, 272, 14611-14616). The WW domain, or in this case the WWP domain, is believed to mediate protein-protein interactions and has been found in several proteins involved in the signaling pathways of growth regulation and cytoskeletal organization that underlie normal and disease processes (Lu et al., Science, 1999, 283, 1325-1328; Rotin, Curr. Top. Microbiol. Immunol., 1998, 228, 115-133; Sudol, Prog. Biophys. Mol. Biol., 1996, 65, 113-132).
WWP1 (also known as Nedd-4-like ubiquitin protein ligase-WWP1, and atrophin-1 interacting protein 5 or AIP5) and WWP2 (also known as Nedd-4-like ubiquitin protein ligase-WWP2, and atrophin-1 interacting protein 2 or AIP2) each contain four WW domains and have been shown to interact with other proteins through these domains. Disclosed in the PCT publication WO 97/37223 are the nucleic acid and polypeptide sequences of WWP1 and WWP2 as well as the sequences of the WW domains encoded within (Pirozzi et al., 1997). Also disclosed are purified nucleic acids that hybridize to WWP1 and WWP2 as well as methods and kits used to identify WW domain-containing proteins.
Wood et al. demonstrated that both WWP1 and WWP2 interact with atrophin-1, the gene product of the DRPLA (Dentatorubral and Pallidoluysian Atrophy) gene (Wood et al., Mol. Cell. Neurosci., 1998, 11, 149-160). DRPLA is a member of a family of progressive neurodegenerative disorders caused by polyglutamine-encoding CAG trinucleotide repeats, and it is the expansion of a CAG repeat in the atrophin-1 gene that specifically results in DRPLA. Investigations of the binding properties of WWP1 and WWP2 with atrophin-1 suggest that WWP1 and WWP2 may play a role in ubiquitin-regulated degradation of atrophin-1. It has also been postulated, based on the similarity between atrophin-1 and huntingtin, a protein shown to bind ubiquitinating enzymes in Huntington's Disease, that WWP1 and WWP2 may interact with huntingtin as well.
Both WWP1 and WWP2 are ubiquitously expressed in all tissues including the nervous system with a particularly high level of WWP1 in the heart and skeletal muscle (Wood et al., Mol. Cell. Neurosci., 1998, 11, 149-160).
In hematopoietic tissues, it has been shown that WWP1 interacts with the DNA-bound transcription factor NF-E2, important for globin gene expression (Mosser et al., Biochemistry, 1998, 37, 13686-13695). NF-E2 is a heterodimeric transcription factor consisting of a hematopoietic-specific subunit and a ubiquitously expressed subunit. It is the hematopoietic-specific subunit of NF-E2 to which WWP1 binds and, based on functional studies of this interaction, it is believed that WWP1 may act as a transcriptional coactivator.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of either WWP1 or WWP2. In light of their proposed association with proteins arising in diseased states, namely atrophin-1 in DRPLA and huntingtin in Huntington's Disease, there remains a long felt need for agents capable of effectively inhibiting the function of these genes.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of WWP1 and WWP2 expression. The present invention provides compositions and methods for modulating WWP1 and WWP2 expression.